Project Summary/Abstract Enzymes that use coupled di-iron clusters that are coordinated by carboxylate and histidine residues to activate dioxygen for difficult oxidation reactions play crucial roles in the global carbon cycle, in DNA biosynthesis, and in synthesis of clinically used natural- product drugs. In this last year, a new structural family of diiron enzymes has come into focus. Related in structure to heme-oxygenase (HO), these HO-like diiron oxidase and oxygenases (HODOs) have already expanded the known catalytic repertoire of the diiron unit, even with only four members of the new family having been assigned biochemical functions. In their functions, these HO-like enzymes produce antibiotics (the nitroimidazoles), cancer drugs (streptozotocin), and jet fuel. Moreover, they appear to function in a manner that is distinct from the functional paradigm that was established by earlier work on other systems. Rather than remaining as stable cofactors within the HO- proteins scaffolds, they spontaneously degrade, at least in vitro, perhaps as part of a novel modus operandi that eliminates the requirement for cooperating proteins in their catalytic cycles. The goal of this project is to understand the structures and mechanisms of the first four functionally assigned members of what appears, on the basis of bioinformatic analysis, to be a large and versatile new enzyme family. The expectation is that an understanding of its functional principles might enable the new family to become a privileged scaffold for directed evolution of new synthetically useful enzyme activities.